The proposed research project focuses on unresolved issues of thrombin allostery that is at the basis of the procoagulant, prothrombotic and anticoagulant functions of the enzyme in the blood. The project builds upon developments from previous NIH support and consists of the following specific aims: 1. map the pathway of long-range allosteric communication between exosite I, the active site and the Na+ site;2. Characterize the E*, E and E:Na+ forms of meizothrombin;and 3. Probe the strategy for macromolecular substrate recognition at the active site that leaves exosite I free for allosteric regulation. In specific aim 1, we will test by site-directed mutagenesis the pathway of long-range communication between exosite I, the active site and the Na+ site identified recently by X-ray crystallographic studies. The contribution of critical residues in this pathway will be probed with Ala or Pro substitutions to test the hypothesis that the mutant forms are stabilized in the E* form and disrupt the way exosite I communicates with the active site and the Na+ site. Functional studies using rapid kinetics and calorimetry will be complemented by X-ray crystallography of selected mutants. These studies will provide much needed information on the energetic and structural contribution of the long-range communication among critical domains of the enzyme. In specific aim 2, we will extend our investigation of thrombin allostery to meizothrombin to test the hypothesis that the E*, E and E:Na+ forms are already present in the most important intermediate along the prothrombin activation pathway. Kinetic, thermodynamic and X- ray structural studies will be performed and mutants of particular importance to thrombin allostery like D102N, W141A, G142P, N143P and Y225P will be expressed and characterized. The X-ray crystal structures of human meizothrombin in the E*, E and E:Na+ will be solved for the first time. These studies will provide important new information on the structure and function of meizothrombin, thereby broadening our understanding of how thrombin allostery ensues along the prothrombin activation pathway. In specific aim 3, we will test the generality of the peculiar fold recently identified for murine PAR4 bound to thrombin that enables the cleaved form of PAR3 to bind to exosite I and act as an allosteric effector. We will use site-directed mutagenesis of PAR4 and X-ray structural biology of human thrombin bound to fragments of PAR1, PAR3, PAR4 and protein C. These studies will reveal much needed information on the key physiological interactions of thrombin with protein C and PARs. PUBLIC HEALTH RELEVANCE: Recent statistics indicate that cardiovascular disease and its thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity in the US and worldwide well into the year 2020. Because of its involvement in thrombotic deaths, thrombin remains a major target of antithrombotic and anticoagulant therapies. Progress in the understanding of thrombin allostery, a regulatory phenomenon at the basis of the procoagulant, prothrombotic and anticoagulant functions of the enzyme in the blood, is therefore a top medical and social priority and has the potential to influence the life-style and life expectancy of millions of people in the US and worldwide.